In the current study, we have shown for the first time how FoxM1/ERK axis affects metastasis in 4T1 cells and its allograft BALB/c mouse model. For this purpose, we used THIO as a FoxM1 specific inhibitor and MEK inhibitor SEL. The possible mechanisms of action of inhibitors, alone or in combination, on metastatic processes were evaluated.
Thiostrepton is an antibiotic derived from Streptomyces azureus that shows significant inhibition of cell proliferation against cancer cells using a combination of direct and indirect inhibition of the transcriptional activity of FoxM1 [19, 20, 35]. In previous studies, 4–10µM concentrations of THIO were reported to adequately inhibit FoxM1 protein expression in different cancer cell lines [20, 36]. Consistent with the literature, our results showed that 4µM concentration of THIO reduced FoxM1 protein expression in vitro. We have given THIO to mice at a lower dose than reported in previous studies. Siraj et al. used 40 mg/kg, whereas Tan et al. used 50 mg/kg THIO dose in a MDA-MB-231 xenograft animal model [21, 37]. Our study demonstrated that 25mg/kg THIO was found to be effective and sufficient to suppress FoxM1 protein expression on the 4T1 allograft breast cancer mouse model.
It was demonstrated that FoxM1 is one of the upstream effectors on the ERK signaling pathway in hepatocellular carcinoma and regulating the ERK signal in breast cancer cells [38, 39]. Furthermore, FoxM1 is activated by cyclin-CDK and ERK-mediated phosphorylation. Phosphorylation of FoxM1 by ERK and p38 from the serine at 331 and 704 positions or other possible regions stimulates the nuclear translocation of FoxM1 [40, 41]. In our in vitro and in vivo treatments MEK inhibitor Selumetinib suppressed ERK phosphorylation (p-ERK) sufficiently. It was also observed that 4 µM Thiostrepton inhibited p-ERK at 24 h in vitro, however 25 mg/kg THIO did not change p-ERK protein expression in vivo.
We have obtained considerable findings on the proteins involved in the epithelial-mesenchymal transition (EMT) which is one of the crucial processes of metastasis. E-cadherin, an epithelial marker in EMT, was increased especially in SEL treatment groups in vitro and in vivo. Previously, p-ERK was reported to be a negative regulator of E-cadherin which is supported by a positive correlation between loss of E-cadherin and ERK activation [42, 43]. Wierstra demonstrated that the DNA-binding domain of FoxM1c binds to the murine and human E-cadherin promoters in vitro . Contrary to this finding, loss of E-cadherin is observed in many cancer cells with increased FoxM1 expression. Park et al. explained this discrepancy with the predominance of Snail-mediated inhibition of E-cadherin expression in tumor cells . However, our in vitro and in vivo findings suggest that E-cadherin expression was mainly affected by ERK inhibition.
We showed that Vimentin, as a mesenchymal marker that supports invasive features in the EMT process, decreased significantly in THIO treatment groups. According to our in vitro study, Vimentin expression decreased in 4 µM THIO and combined treatments in 6 and 24 h, while SEL treatment did not change Vimentin expression. Therefore, we can conclude that the decrease observed in the combined treatments may be due to the effect of FoxM1 inhibitor THIO. There are studies showing that reduced Vimentin expression in FoxM1 knock-out (KO) mouse models or after silencing FoxM1 expression in various human cancers [15, 45]. In a study investigating the role of FoxM1 in the EMT process in hepatocellular carcinoma, it was reported that Vimentin expression increased with the FoxM1 overexpression, that Vimentin was a target of FoxM1 directly or indirectly . Therefore, in our study, the decrease in Vimentin expression in THIO treated groups could be associated with FoxM1 inhibition. The exact molecular mechanism of THIO on Vimentin should be investigated in further studies.
Twist protein expression, which is another marker of epithelial-mesenchymal transition and metastasis, was found to be suppressed both in vitro and in vivo in all treatment groups. Qian et al. showed that the human FoxM1 gene promoter region contains a binding site for the Twist protein, and can be uniquely linked to the FoxM1 promoter of Twist by ChIP analysis. Thus Twist has been reported to be a direct transcriptional target of FoxM1 . Besides, Twist's largest phosphorylation region was stated to be phosphorylated by ERK from serine 68, thereby maintaining its stability but its expression decreased during the inhibition of MAPK activities . In this context, together with FoxM1 and MEK inhibition, could have a strong effect in suppressing Twist expression which is the master regulator of tumor metastasis.
The expression and activity of the matrix metalloproteinases (MMP) increase in almost all types of breast cancer and is associated with advanced stage of the tumor and poor prognosis [49, 50]. The expression of MMP-2 and MMP-9 proteins isolated from our in vitro experiments could not be determined by the Immunoblot method. Although MMPs are connected with survival and expansion of cancer cells, they are produced in very small quantities . Cancer cells in a paracrine manner, stimulate tumor microenvironment to supply sufficient MMPs from them . This phenomenon may explain why we could observe the expression of MMP-2 and MMP-9 in protein samples isolated from tissues, but could not observe in protein samples isolated from in vitro experiments. Our in vivo findings demonstrated that MMP-2 expression was decreased significantly in all groups as compared to the control, whereas MMP-9 expression was decreased only in THIO and combined treatment groups. Wang et al. (2007) reported that a decrease in FoxM1 expression reduced MMP-2, MMP-9 and VEGF expression in pancreatic cancer cells, thereby reducing the metastasis ability of these cells . It is demonstrated that FoxM1 increases its activity by directly connecting to MMP-2, and indirectly regulates MMP-9 via downstream target of JNK1.
Malignant tumors have the capacity to break down the extracellular matrix (ECM) with controlled proteolysis. One of the proteolytic systems that play a role in these processes is the urokinase type plasminogen activator (uPA) system. Studies have demonstrated that the expression of FoxM1 is positively correlated with urokinase-type PA(uPA), MMP-2 and MMP-9 expressions [55, 56]. Previously, Ma et al reported that overexpression of FoxM1 and the uPA system were associated with gastric cancer progression and poor prognosis . We evaluated uPA activity in mice plasma using an Elisa kit and found a significant decrease in uPA level only in the THIO group. According to the findings of this study, the decreases in the level of uPA, MMP-2 and MMP-9 protein expression via THIO confirm that FoxM1 regulates ECM proteolysis through this system.
In this study, a wound healing assay was carried out to evaluate cell migration of 4T1 cells. According to our results, while both concentrations of THIO significantly inhibited cell migration, only 16µM concentration of SEL caused a decrease in cell migration. We also found a significant reduction in cell migration in combined treatments. These results are consistent with our in vitro findings, such as the increase in E-cadherin protein expression, decrease in Vimentin protein expression, and decrease in Twist expression, which are the markers that encourage cell motility.
4T1 mouse breast cancer cells are highly invasive, so they can spontaneously form metastases . When 4T1 cells are implanted into BALB/c mice, they spread to distant organs such as the lung, liver, brain . In our allograft breast cancer tumor model, the percentage of metastasis in the control group was 57.14% in the control group, whereas this ratio decreased in THIO (33.33%) and the combined group (40%). THIO may have reduced distant organ spread by altering protein expression associated with metastasis. Administration of mice 25 mg/kg THIO to the mice every other day decreased tumor volume significantly, however no significant decrease in tumor size was observed in the combined group. We observed that the combined group of mice has the shortest survival rate. Despite its promising effects at the molecular level on metastasis, we suggest that THIO should be followed up with more groups of mice and prolonged days to analyze survival and tumor growth.
As a conclusion, our study revealed that FoxM1 inhibitor THIO alone was more promising on proteins involved in metastasis steps as compared to SEL-alone treatment. However, both inhibitors affected EMT with different molecular processes and that their combined usage had a stronger effect on EMT. THIO is an FDA-approved antibiotic used for the treatment of dermatological diseases in veterinary applications. However, it has no application for human use. The difficulty of its synthesis, degradation potential and its hydrophobic form make it difficult to use in the clinic. Therefore, in order to overcome these problems, we think that THIO should be formulated in nanoparticle or microparticle drug delivery systems and should be investigated in vitro and in vivo considering the toxicological and genotoxic effects of these formulations.